SUMMARY OF WORK This research area involves a model of in vitro differentiation of cardiomyocytes originating from embryonic stem cells (R1). The research is aimed at generating cardiac-lineage specific cells to understand the role of proteins important for excitation-contraction-relaxation coupling and differentiation processes leading to the formation of cardiomyocytes in vitro. We have been able to successfully differentiate pluripotent ES cells into embryoid bodies containing contracting cardiac-like cells. Immunofluorescence analyses of the differentiating ES-derived cardiac cells using monoclonal and polyclonal antibodies have been successfully performed for sarcomeric actins, troponin T; specific work on identifying the earliest stages of ryanodine receptor, SR CaATPase, phospholamban and dihydropyridine receptor expression have also been successful. We have established several techniques to measure the mRNA contents of these proteins, all of which are being used to develop a molecular model for the development of EC coupling and relaxation in early and late differentiating ES cells. Using puromycin resistance cassettes with cardiac-restrited promoters, we have generated a number of ES cell lines that, when differentiated, can be used, at least partially, to select for ventricular-specific cell lineages. We have also developed a number of ES cell clones with a targeted the mouse ryanodine receptor 2 (RyR2) gene which have been utilized with Cre Recombinase to introdude of loxP flanking cDNA constructs. Using these cells, we have co-transfected cDNA constructs containing puromycin resistance cassettes flanked by loxP sites with an expression vector for Cre Recombinase. After cotransfection, positive selection techniques have been used to identify clones where the targeted RyR2 alleles have been replaced by puromycin resistance cassettes. This demonstrates that floxed RyR2 exon can be successfully deleted and that a puromycin resistance cassette can be inserted into the same locus at high efficiency. New constructs are now being prepared to form chimeras between the endogenous mouse gene and a human cDNA construct.This system will ultimately allow us to rapidly create chimeric mutants for RyR2 so that structure-function characteristics of this protein can be studied on isolated cells. A number of the clonal cell lines have now been partially characterized. The results with the RyR KO cells indicate that the ryanodine receptor is not necessary for the differentiation of beating cardiac myocytes from ES cells, but that it plays a critical role in the regulation of the beating rate of cardiac myocytes. Additionally, we have used genomic techniques like serial analysis of gene expression or microarrays to identify a number of transcripts that may be involved in the differentiation processes to cardiomyocytes. These gene targets are the subject of active investigation. The aim is to identify factors that will promote the development of cardiomyocytes in vitro, or which can be utilized to lead to repair of the damaged heart in vivo.